Mosquitoes are vectors of numerous infectious diseases. To foster the development of advanced genome- based strategies for vector control, over thirty species have been sequenced and their genomes made publically available through the NIAID funded Bioinformatics Resource Center (BRC) VectorBase. The genome assembly of Culex quinquefasciatus, the major vector of the lymphatic filariasis worm and encephalitis viruses, however remains the most highly fragmented of the major mosquitoes with only 13% of its genome placed onto chromosomes. The lack of a high-quality, chromosome-based genome assembly for Cx. quinquefasciatus remains a substantial impediment to further progress in Culex biology and vector comparative genomics. Using PacBio sequencing, for which we have already assembled two Anopheles genomes de novo, we will reassemble Cx. quinquefasciatus and produce a significantly more contiguous assembly aided by both novel computational innovations and traditional physical mapping. The total number of scaffolds (pieces, also called supercontigs) will be reduced from 3171 to at most ~1750. In addition, this increase in assembly continuity will greatly augment the current cytogenetic map for Cx. quinquefasciatus and help anchor the majority of the genomic scaffolds (old, new or hybrid) to chromosomes. Although the primary goal of this R21 proposal is to significantly improve the genome assembly for Cx. quinquefasciatus, we undertake it with the long-term goal of piloting the sequencing and bioinformatics required to improve other important vectors (e.g., ongoing sandfly and black fly community efforts). The specific aims for this project are to: 1) improve the quality of the Cx. quinquefasciatus genome assembly through PacBio sequencing; 2) augment and improve this assembly using multicolor fluorescent in situ hybridization; and 3) innovate assembly improvement and comparison within and across mosquitoes. The availability of a high quality genome assembly for Cx. quinquefasciatus through VectorBase will greatly enhance research in vector biology.